Field of the Invention
The present invention relates to use of inducible promoters in the production of methionine by fermentation.
Background of the Invention
Sulphur-containing compounds such as cysteine, homocysteine, methionine or S-adenosylmethionine are critical to cellular metabolism and are produced industrially to be used as food or feed additives and pharmaceuticals. In particular methionine, an essential amino acid, which cannot be synthesized by animals, plays an important role in many body functions. Aside from its role in protein biosynthesis, methionine is involved in transmethylation and in the bioavailability of selenium and zinc. Methionine is also directly used as a treatment for disorders like allergy and rheumatic fever. Nevertheless most of the methionine that is produced is added to animal feed.
With the decreased use of animal-derived proteins as a result of BSE and chicken flu, the demand for pure methionine has increased. Chemically D,L-methionine is commonly produced from acrolein, methyl mercaptan and hydrogen cyanide. Nevertheless the racemic mixture does not perform as well as pure L-methionine, as for example in chicken feed additives (Saunderson, C. L., (1985) British Journal of Nutrition 54, 621-633). Pure L-methionine can be produced from racemic methionine e.g. through the acylase treatment of N-acetyl-D,L-methionine which increases production costs dramatically. The increasing demand for pure L-methionine coupled to environmental concerns render microbial production of methionine attractive.
Use of inducible promoters in biotechnological processes is in the art of chemical biosynthesis. These promoters usually respond to chemical or physical stimuli exemplified by propionate (WO2007005837), zinc (WO2004020640) and arabinose (WO1998011231) or temperature (Microbial conversion of glycerol to 1,3-propanediol by an engineered strain of Escherichia coli. Tang X, Tan Y, Zhu H, Zhao K, Shen W. Appl Environ Microbiol. 2009 March; 75(6):1628-34.) and light, respectively.
Methionine production relies on several precursor providing pathways. Efficient methionine production requires fine tuning of these pathways. For maximum methionine production it can be beneficial to be able to modulate the expression of certain key enzymes during the process. For example (i) the expression of certain enzymes is only required during the production phase and not during the generation of the biomass or vice versa. Other enzymes are only beneficial in stationary phase. Therefore, use of inducible promoters may be of interest in improving the overall yield of producing methionine at an industrial level.
However, due to the complexity of the methionine metabolic pathway and the fine tuning of these pathways for an improved methionine production, use of inducible promoters to control expression of genes involved in methionine production was never considered and reported.
The inventors have found now that inducible promoters may be beneficial when used to regulate gene expression of genes involved in complex metabolic pathways such as methionine biosynthesis.